| During the culture of the Chinese mitten crab(Eriocheir sinensis) in China, trashfish and fish meal is usually fed as food or feedstuffs, which decays rapidly, due toimproper processing such as long-distance transport, high temperature of fish mealpreparation, bad condition of storage and so on. However, stale trash fish contain highlevel of biogenic amines, such as histamine. The presence of this compound in foods orfeedstuffs at high levels is considered to be toxic and ingestion causes effects such assurvival, decreased weight gain and delayed sexual maturity in the mysis.Therefore,histamine is used as a quality criterion for fish meals and also as an important safetyindicator for food. At present, dietary histamine supplementation caused intestinaldamage of rainbow trout, but no further information about digestive function can befound in crustacean.The aim of the current study on mysis shrimp was to providemethod pattern on the E. sinensis for further research.It is the first time to report on theeffect of histamine in the digestive function, immune and development of ovary incrustacen.The results was listed below:(1) The formulated diets containing different levels of supplemented histamine (0,1000,2000and4000mg/kg) were fed to mysis shrimp(Neomysis japonica) for17d andE. sinensis for28d. Hepatopancreas(Hp) and intestine were used to analyse thechanges of histological structure and digestive enzyme activity.a) The results showed that histamine caused the severe loss of regular structure andsloughing of epithelial cells(1000mg/kg); the increase of B cells and thinness tubularwall(4000mg/kg) in Hp of mysis shrimp and Chinese mitten crab; the increase of Fcells and bulges of tubler wall(2000mg/kg) in the Chinese mitten crab.b) There were notable structural alterations of the intestine including epithelial cellsarranged in sparse, separation of necrotic cell from basal laminae(1000mg/kg), thinedhindgut wall and the sloughing off of the intestine film (4000mg/kg) in mysisshrimp;while no similar influence in E. sinensis.Histamine led the hindgut ampulla ofthe juction with midgut and hindgut less basophilic,and reticulate structure less eosinophil(1000mg/kg), the increase of basophilic and eosinophil materials in theampulla hindgut and reticulate structure respectively (2000and4000mg/kg), thereduction of eosinophil materials in the connective tissue of hindgut(2000and4000mg/kg) and the increase of eosinophil materials in the ridge of thehindgut(1000mg/kg).c) PCNA-immunoreactivity(PCNA-ir) was present in the whole intestine.Theintensity of PCNA-ir in the hindgut ampulla was similar to those of basophilicmaterials, the PCNA-ir in the ridge accord to those of eosinophil materials in differentlevels of supplemented histamine groups.Those basophilic and eosinophil materials inthe hindgut ampulla and hidgut were strongly colored by alcian blue/safranin O andToluidine blue (TB).The observation that basophilic materials in the hindgut ampullaand midgut also displayed histamine positive.(2) Histamine reduced the digestive enzyme activities of Hp and intestine,especially in the Hp.a) Trypsin and lipase of Hp were significantly lower in the1000mg/kg histaminegroup than the control on the7,14,21and28d(p<0.05)；amylase of Hp wassignificantly lower than the control on the7and14d(p<0.05). Trypsin, amylase andlipase of the intestine were significantly lower in the1000mg/kg histamine group thanthat in the control on7d,7d~21d and28d respectively.b) Among the three digestive enzymes of the Hp, the trypsin was effected byhistamine dramatically at each time point. On the7d, the activities of trypsinsignificantly decreased to27.00%(1000mg/kg)、71.79%(2000mg/kg)、57.75%(4000mg/kg) compared with the control group (1.76±0.02)U/(mg.prot)(p<0.05). On the14~28d,the activity of the trypsin was inhibited by all levels of histamine(p<0.05), except4000mg/kg on the28d. Amylase reduced on the7d and14d(p<0.05), but there was nodifference between histamine-treated and the control groups on the21and28d(p>0.05).On the14,21d and28d, lipase only in the group of1000mg/kg histaminewas decreased significantly compared with the control(p<0.05).c) Quantitative real-time RT-PCR(qRT-PCR) was employed to investigate theexpression of trypsin mRNA in different levels of histamine in Hp of crabs, the resultsshowed that the expression was in according with the trypsin activities.(3) Histamine temporally accumulated in the muscle and the Hp of the crab at96h after injection with0,1,50,100and200g histamine g-1crab-1, further more histamine concentration was positively correlated to the dose of injected histamine.The concentration of5-HT and octopamine in the Hp and muscle were increased withhigh levels of histamine(0,50,100and200g histamine g-1), but putrescine showed notrend. The crab fed0,1000,2000and4000mg histamine kg-1of diet and tested thehistamine concentration in a feeding trial for28days. The histamine concentration inthe Hp of histamine-treated groups was elevated significantly compared with thecontrol on the7d(p<0.05), but no histamine can be deteceted in the muscle. DAO inthe Hp, serum and muscle of the crab after feeding histamine trial on the28d wasmeasured. The DAO activity was the higest in the muscle and lowest in the serum.(4) Compared with Neomysis awatschensis, Neomysis japonica was moresusceptible to Vibrio alginolyticus, and had a higher death rate than that of Neomysisawatschensis in all the histamine concentrations at96h. Heamocytes of Neomysisawatschensis included the higher ratio of granular cells and more various morphology.According to light and electron microscopical studies, three types of haemocytes wererecognized in N.japonica: hyaline cells(no cytoplasmic granule), semigranular cells(afew cytoplasm granule), granular cells(abundant granules in cytoplasm). Theproportion of granular cells (47.53%±0.02) was the most, while that of hyalinocytes(21.24%±0.02) the least. The size was increased from hyaline cells to granular cells,however, the N/C rate was decreased. N. japonica were exposed to variousconcentrations of histamine(10mg/L,20mg/L and40mg/L) for30d and the rate ofgranular cells(p<0.05)and the density of cytoplasmic granules was decreased markedly.At histamine dose of40mg/L, the morphology of granular became more various.Theactivites of acid Phosphatase(ACP), alkaline Phosphatase(AKP), lysozyme(LYZ), andphenoloxidase (PO) were analyzed in total body of N.japonica.We found that LYZ andPO were decreased with the level of histamine elevated(p<0.05) and had no differencein the ACP and AKP (p>0.05).The crabs infected with the common bacteria pathogen Aeromonas hydrophilawere injected with0,1,50or100g histamine g-1crab-1and their survivorship after96h was recorded. The results showed that histamine alone had no influence on thecrab survival but can decrease the survival of the crabs challenged with A. hydrophilaafter36h. Additionally, the survival of the crabs injected with the high level (100μgg-1) histamine was significantly higher than that of the lower level (50μg g-1) after60hto96h(p<0.05), but not different from the control. Immune parameters of the hemolymph in the crabs, such as the total hemocyte counts (THC) and thephenoloxidase(PO), acid phosphatase(ACP), alkaline phosphatase(AKP) and superox--ide dismutase activity (SOD), were measured at6,12and24h after injection withhistamine. Some immune parameters, such as PO and SOD, showed an increase, whileTHC, ACP and AKP levels declined at6h. Surprisingly, after the injection ofhistamine, the crabs quickly released their limbs and began to twitch, and the numberof the crabs with autotomy and spasm increased with the increasing histamineconcentration(p<0.05). It is the first time to observe that histamine could lead the crabautotomy. Overall results of this study indicate that the regulation of histamine in thesurvival of E. sinensis resistance against A. hydrophila and immune parameters dependdirectly on the concentration of histamine.(5) According the study on immunolocalization of estrogen receptor-α inNeomysis japonica oocytes during ovarian development, we detect for the first timethe ERα in oocytes, follicle cells and hepatopancreas cells of mysis byimmunohistochemistry using a specific ERα antibody. ERα was mainly localized in thenuclei of oocytes and follicle cells, while mainly detected in nuclei of oogonia (OG),previtellogenic oocyte (PR) and endogenous vitellogenic oocyte (EN) at previtellogenicand early vitellogenic stage (I-early III).Therefore, in E. sinensis we choosed theovarian cycle(stage I) to research the effect of histamine on the ovarian development.After96h injected histamine at dose of0,1,50,100and200g/g, the ovary wasdissected for histological studies. The observation exhibited that histamine led theintercellular space expand and the regular form of oocytes lose. After histamineinjection at dose of50g/g, the ovary arranged in sparse widely and the oocyte in thecentre of the ovary showed a marked atrophy, what’s worst, the cavity replaced theshrinking oocyte. The histological damage of histamine-injected crabs at dose of100g/g was similar with that of50g/g. After200g/g histamine treated, the oocytesat margin of ovary produced many small granule cells. By immunohistochemistry,PCNA-ir was intense in the irregular oocytes and small granule cells, while theintensity of ER-ir in the oogonia, regular oocytes(PR and EN) and small granule cellwas stronger than the shrinking oocytes. |
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